猫PD-L1单克隆抗体及猫源化单链抗体的制备和初步应用

doi:10.11843/j.issn.0366-6964.2026.01.035

Abstract

Abstract:

The purpose of this study was to prepare feline PD-L1 antigen and screen monoclonal antibody， and preliminary application was carried out in detection and diagnosis of canine and feline oncology diseases. In addition， feline derived chimeric single chain variable Fragment-Feline Fragment crystallizable （scFv-fFc） was prepared， which provided a tool for the treatment of cat tumor in future. The PD-L1 gene of feline was amplified by PCR and prokaryotic expression recombinant plasmid PET-MPD-L1 was constructed， and the antigen of PD-L1 was prepared by expression with host BL21（DE3）. After immunization with mice， cell was fused， and an ELISA was established to screen antibody produced by hybridoma cells. After subcloning three times， the cell line of MPD-L1 hybridoma were established， subtypes were identified， and the ascites were prepared， and titer of the ascites was determined. Characteristics were determined by Western blot and immunohistochemistry （IHC）. Total RNA was extracted from the hybridoma cells， and the heavy chain variable region （VH） and light chain variable region （VL） genes were amplified by RT-PCR， which were compared with the antibody light and heavy chain genes in the IMGT database for confirmation. RNA from peripheral blood of a cat in clinic was extracted， and the Fc gene of IgG1 was amplified by RT-PCR. The recombinant plasmids of pFast-MPD-L1-scFv and PFAST-MPD-L1-scFv-fFc were constructed by splicing VH and VL into scFv and scFv-Fc into scFv-fFc by landing PCR， then expressed in the baculovirus expression system. The results showed that feline PD-L1 was highly expressed in the supernatant of cultures and the purified protein could be used antigen for immunization. The monoclonal antibody of 2A5A2F4D7 belonged to IgG 2a subtype， and the light chain is subtype κ. The titer of the supernatant， ascites was 1：3200， 1：102400 respectively. Western-Blot results showed that monoclonal antibody 2A5A2F4D7 reacted with expressed feline PD-L1， and IHC results showed that the monoclonal antibody secreted by 2A5A2F4D7 hybridoma cells can recognize PD-L1 of tumor cells both canine and feline. The VH and VL genes amplified were consistent with genes in the IMGT database， with 4 framework regions and 3 hypervariable regions. The recombinant plasmids of pFast-MPD-L1-scFv and PFAST-MPD-L1-scFv-fFc were constructed successfully and expressed in the supernatants of insect baculovirus expression system. The prepared feline PD-L1 monoclonal antibody have certain value in the detection and prognosis of canine and feline tumors， while scFv-fFc might hold great potential for application in the treatment of tumors in canine and feline.

Key words: tumor diseases in canine and feline, feline PD-L1, monoclonal antibodies, scFv, scFv-fFc, insect baculovirus expression

CLC Number:

S856

LI Meng, SU Siyuan, LI Zipan, XU Yuntiao, HUANG Zibei, LUO Jianya, ZHAO Yiheng, GUO Xin, BIAN Hongkai, PAN Xinyu, LIU Wenbo. Preparation and Application of Cat PD-L1 Monoclonal Antibody and Feline Derived Single Chain Antibody[J]. Acta Veterinaria et Zootechnica Sinica, 2026, 57(1): 401-413.

Figures/Tables 11

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Preparation and Application of Cat PD-L1 Monoclonal Antibody and Feline Derived Single Chain Antibody

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